Sialidase localized in plasma membrane and DNA coding for the same

ABSTRACT

Sialidases localized on plasma membranes having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and DNA coding for the sialidase having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

This application is a national stage PCT/J98/02072, filed May 11, 1998.

TECHNICAL FIELD

The present invention provides a novel sialidase and DNA coding for it.More precisely, the present invention provides sialidase that localizesin plasma membrane, and specifically hydrolyzes gangliosides, and DNAcoding for it.

The sialidase of the present invention and the DNA coding for it areexpected to be utilized as a reagent used for saccharide chain studiesand a medicament used for gene diagnosis and gene therapy.

BACKGROUND ART

Sialidase is a glycohydrolytic enzyme present in living bodies, whicheliminates a sialic acid residue from a non-reducing terminal ofsaccharide chains of glycoproteins or glycolipids. It has been knownthat, when sialic acid is removed from saccharide chain molecules, notonly the degradation of these molecules begins, but also molecularconformation and many of important cell functions such as recognitionmechanism by receptors, cell adhesion, and immunomechanism may bechanged. It has also become clear that sialidase exhibits rapid activitychange in connection with proliferation and canceration of cells, and itis involved in the metastatic ability of cancer cells. However, there islittle knowledge about how sialic acid is eliminated in vivo. This isbecause the studies of mammalian sialidases on the molecular level arebehindhand, and hence there are many unknown points concerning theirstructures and expression mechanism.

Because mammalian sialidase exhibits only low activity, and is extremelyunstable, isolation and purification of the enzyme have been difficult.Sialidase has been often considered for a long time to be one of themere lysosomal enzymes involved in the dissimilation and degradation.Under such a situation, we isolated and purified the enzyme by usingmainly rat tissues as the source of the enzyme, and found that therewere four types of sialidases which differ from sialidases of bacteria,viruses, protozoa and the like in their natures (Miyagi, T. and Tsuiki,S., Eur. J. Biochem. 141, 75-81, 1984; Miyagi, T. et al., J. Biochem.107, 787-793, 1990; Miyagi, T. and Tsuiki, S., J. Biol. Chem. 260,6710-6716, 1985). These enzymes each localize in lysosomal matrix,lysosome membrane, plasma membrane (cell surface membrane), andcytoplasm within a cell, respectively, and they are different from eachother not only in enzymological characteristics such as substratespecificity, but also in immunological properties. Among thosesialidases, the sialidase localized in cytoplasm can be obtained as ahomogenous purified product from rat skeletal muscles. Its cDNA cloninghas been succeeded for the first time in the world as for animalsialidases, and its primary structure has been determined (Miyagi T. etal., J. Biol. Chem., 268, 26435-26440, 1993). Its genomic structureanalysis has also been done, and as for its function, it has beenelucidated that the enzyme participates in the differentiation and thegrowth of skeletal muscle cells by using the cDNA as a probe. Thesestudies can be considered a part of pioneer researches in sialidasestudies in the world.

By the previous studies, it has become clear that there is possibilitythat the sialidase localized in plasma membrane exhibits activityelevation upon proliferation and canceration of cells, and it is alsodeeply concerned with the differentiation of nerve cells and the signaltransduction of cells. To date, however, it has not been understood atall about the structure of this enzyme, the mechanism causing theactivity change and the like. In order to answer these questions, whatmany researchers in this field have long been desired is cloning of itscDNA. For example, if the mechanism of cancerous change due to thisenzyme could be elucidated, it would be possible to utilize the resultsin diagnosis and therapy of cancers. Further, because gangliosides existin surface membranes of many cells and participate in important cellfunctions such as cell adhesion and informational communication, andthey are also main cerebral components, the sialidase utilizing them asa specific substrate is estimated to be involved in certain importantcranial nerve functions.

SUMMARY OF THE INVENTION

The present invention has been accomplished in view of theaforementioned present condition. An object of the present invention isto provide the sialidase localized in plasma membrane and DNA that codesfor it.

The inventors of the present invention earnestly conducted studies inorder to achieve the aforementioned object, and as a result, succeededin isolating the sialidase localized in plasma membrane and cloning ofcDNA coding for it. Furthermore, they found that the aforementionedsialidase was unique in that it substantially specifically hydrolyzedgangliosides (glycolipids containing sialic acid), which are substratesthat similarly localize mainly in plasma membrane, and it was completelydifferent from other mammalian sialidases and microbial sialidases inenzymatic substrate specificity. Thus, they accomplished the presentinvention.

That is, the present invention provides a protein defined in thefollowing (A) or (B):

(A) a protein which has the amino acid sequence of SEQ ID NO: 2 or SEQID NO: 4, or

(B) a protein which has the amino acid sequence including substitution,deletion, insertion, or transition of one or several amino acid residuesin SEQ ID NO: 2 or SEQ ID NO: 4, and exhibits activity to eliminate asialic acid residue from a non-reducing terminal of ganglioside.

The present invention also provides DNA coding for the protein definedin the above (A) or (B). Specifically, such DNA may be DNA which has thenucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

A sialidase which has the aforementioned characteristics will bereferred to as the “sialidase of the present invention” hereinafter, andDNA coding for it will be referred to as the “DNA of the presentinvention” hereinafter.

Hereafter, the present invention will be explained in detail.

<1> Sialidase of the Present Invention

The sialidase of the present invention is a protein which has the aminoacid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Moreover, the sialidaseof the present invention include a protein which has the amino acidsequence including substitution, deletion, insertion, or transition ofone or several amino acid residues in SEQ ID NO: 2 or SEQ ID NO: 4, solong as it exhibits activity for eliminating a sialic acid residue froma non-reducing terminal of ganglioside.

Among the aforementioned sialidases, the sialidase that has the aminoacid sequence of SEQ ID NO: 2 has the following physicochemicalproperties.

(1) Activity

It eliminates sialic acid residues from a non-reducing terminal ofganglioside.

(2) Substrate Specificity

It acts on gangliosides, but not act on glycoproteins andoligosaccharides. Specifically, it acts on GD3-ganglioside,GD1a-ganglioside, GM3-ganglioside, and synthetic gangliosides(GSC-17(α2-3) and GSC-61(α2-6)), but it does not substantially act onGM2-ganglioside, GM1-ganglioside, orosomucoid, fetuin, glycophorin,ovine submaxillary gland mucin, and bovine submaxillary gland mucin. Itweakly acts on α2-3 sialyllactose, and 4-MUNeuAc (4-methylumbelliferylN-acetylneuraminic acid).

(3) Optimum pH

4.7 to 5.0

(4) Molecular Weight

About 65,000 as determined by sucrose density gradient centrifugation,

About 52,000 as determined by SDS-polyacrylamide gel electrophoresisunder reducing condition

(5) Inhibition and Activation

A surface active agent is required for the activity. For example, it ishighly active in the presence of 0.1 to 0.2% of Triton X-100.

It is strongly inhibited by heavy metal ions such as Cu²⁺, and 4-hydroxymercury benzoate.

It is stabilized by dithiothreitol, Neu5Ac2en(2-deoxy-2,3-dehydro-N-acetylneuraminic acid), and glycerol. However, itis weakly inhibited by Neu5Ac2en.

Among the sialidases of the present invention, the sialidase which hasthe aforementioned characteristics is an enzyme derived from bovine,whereas the sialidase which has the amino acid sequence of SEQ ID NO: 4in one derived from human. These exhibit 82% homology in their aminoacid sequences, and they have a transmembrane domain, glycosylationsite, and Asp-box, which is a consensus sequence of sialidase, at thesame locations. Therefore, the enzyme derived from human is consideredto have the same physicochemical properties as the enzyme derived frombovine.

The sialidase of the present invention can be obtained from a bovinebrain, for example, as follows. All of the following procedures arepreferably carried out at a low temperature.

A bovine brain is homogenized in a buffer, and centrifuged at 1000×g for10 minute, and the supernatant is further centrifuged at 30,000×g for 1hour. After the centrifugation, the precipitate fraction is suspended ina buffer, added 5% deoxycholic acid, sufficiently homogenized, andcentrifuged at 100,000×g for one hour to obtain a soluble fraction as asupernatant. The buffer preferably contains an inhibitor for proteases,dithiothreitol, surface active agent and the like.

The above soluble fraction is applied to a DEAE-cellulose column and,after the column is washed, eluted with a buffer containing 0.2 M NaClfor fractionation. A fraction exhibiting the sialidase activity isdialyzed against a buffer, then applied on Octyl-Sepharose, andseparated by elution with a linear gradient of 0.1-0.4% Triton X-100.

Then, an active fraction is applied to Heparin-Sepharose (Pharmacia),washed with a buffer containing 0.25 M NaCl, and eluted with a 0.2-1 MNaCl linear gradient to concentrate the active fraction. The aboveconcentrated enzyme solution is loaded on Sephacryl S-200 (Pharmacia),and separated by elution with a buffer containing 0.02 mM NeuAc2en(2-deoxy-2,3-dehydro-N-acetylneuraminic acid).

The obtained active fraction is diluted to have a Triton X-100concentration of 0.02%, added to RCA-lectin agarose (Pharmacia), washedwith a buffer containing 0.02% Triton X-100, and eluted with a buffercontaining 0.2 M lactose. This active fraction is loaded on a MonoQ(Pharmacia) column, and eluted with a 0-0.5 M NaCl linear gradient.

The active fraction is loaded on an activated thiol Sepharose(Pharmacia) column, washed with a 0.15 M NaCl buffer containing 10%glycerol, and then with 0.5 M NaCl buffer containing 10% glycerol, andeluted with 0.05 M NaCl buffer containing 0.05 M dithiothreitol. Theactive fraction is concentrated in a MonoQ column.

The above concentrate is loaded on an affinity column utilizing asynthetic ganglioside GM3 [GSC-211, NeuAc-Gal-Glc-O(CH₂)₈NH₂] as aligand (Hasegawa A. et al. J. Carbohydr. Chem., 9, 201-214, 1990), andseparated by elution with a 0-0.5 M NaCl gradient. The affinity columncan be obtained by allowing GSC-211 to couple with ECH-Sepharose(Pharmacia) in the presence ofN-ethyl-N′-(3′-dimethyl-aminopropyl)carbodiimide hydrochloride.

The sialidase enzyme is purified as described above as a protein havinga molecular weight of 52 kD as determined by SDS-polyacrylamide gelelectrophoresis.

Further, since DNA coding for the sialidase of the present invention hasbeen obtained, the sialidase of the present invention can also beobtained by expressing the DNA in a suitable host-vector system. As forthe host-vector system, a cultured cell can be used as a host, and avector suitable for this host can be used. Materials and methodstherefore may be those usually used for the production of heterogenousproteins utilizing genetic recombination techniques. When DNA coding forthe sialidase of the present invention is ligated to a vector, a vectorcontaining sequences required for regulation of gene expression such aspromoter and terminator that can be expressed in the host as requiredmay be used.

<2> DNA of the Present Invention

Because the amino acid sequence of the protein encoded by the DNA of thepresent invention has been elucidated, the DNA of the present inventioncan be cloned based on the amino acid sequence. In the examplesmentioned below, a partial amino acid sequence of the sialidase of thepresent invention is determined, oligonucleotide primers are synthesizedbased on the partial amino acid sequence, and the DNA of the presentinvention is obtained from a bovine brain cDNA library by PCR(polymerase chain reaction) using the oligonucleotides primers.

Although the sequence of the DNA of the present invention is notparticularly limited so long as it codes for the amino acid sequence ofSEQ ID NO: 2 or 4, the nucleotide sequences of SEQ ID NOS: 1 and 3 canbe specifically mentioned. Further, existence of sialidases having theamino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 includingsubstitution, deletion, insertion, or transition of one or several aminoacid residues, and genes coding for them is expected due to differenceof animal species, individuals or varieties. Such DNA coding for thesubstantially same protein as the sialidase of the present inventionalso falls within the scope of the DNA of the present invention. SuchDNA can also be obtained from a cell harboring it by hybridizing it withthe nucleotide sequence of SEQ ID NO: 1 or 3 or a part thereof under astringent condition, and isolating DNA coding for a protein which hassialidase activity. DNA coding for a sialidase having such a mutation asmentioned above may also be obtained by, for example, site-specificmutagenesis or mutagenic treatment.

The term “one or several” amino acid residues means 1-80, preferably1-30, more preferably 1-5 amino acid residues.

<3> Pregressive Applications of the Sialidase of the Present Inventionand DNA Coding for It

(1) Because the sialidase of the present invention exhibits substratespecificity unique in that it substantially specifically hydrolyzegangliosides, a recombinant having the enzyme or DNA coding for theenzyme has much possibility for use as a reagent for saccharide chainstudies.

(2) As one of the means for normalizing abnormality of this enzymeobserved in cancer cells, for example, antisense therapy, which is akind of gene therapy, will be expected in future. The gene structureclarified by the present invention is the important information for it.Moreover, if the expression mechanism of this enzyme becomes clear bygenome structure analysis utilizing the cDNA as a probe, it will alsobecome possible to normalize the abnormality of the expression of thisenzyme in cancer and the like.

(3) Because of two reasons, i.e., the characteristic that the enzymespecifically decomposes gangliosides that are main components of brain,and its involvement in differentiation of nerve cells, the abnormalitiesof this enzyme may be found in certain brain diseases. In such a case,the information about the gene may be much utilized for development ofgene therapy and medicaments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the amino acid sequences of peptides obtained byendoproteinase digestion and lysyl endopeptidase digestion. The aminoacids represented with minor characters in the figure are amino acids oflow determinancy. A Lys residue presumed to have bound to the N-terminusamino acid is represented with (K).

FIG. 2 represents alignment of deduced amino acid sequences of a PCRproduct (BBmSD) obtained by using bovine brain cDNA as a template and arat skeletal muscle cytoplasmic sialidase (RMcSD). Common amino acidsare represented with dots “.”, and analogous amino acids are representedwith asterisks “*”. The highly homologous regions used for thepreparation of a probe are underlined.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained more specificallywith reference to the following examples.

<1> Purification of Sialidase Localized in Plasma Membrane

(1) Method for Measuring Sialidase Activity

In this example, sialidase activity was measured as follows.

A reaction system (0.2 ml) contained 50-100 nmol of sialic acid-boundsaccharide substrate, 0.2 mg of bovine serum albumin, 15 mmol of sodiumacetate buffer (pH 4.6), 0.2 mg of Triton X-100, and an enzyme fraction,and a substrate consisting mainly of bovine brain mixed gangliosides(Sigma, Type II) was used.

A reaction mixture having the aforementioned composition was incubatedat 37° C. for 15-60 minutes, and the reaction was stopped by quickfreezing. The released sialic acid was quantitated by the thiobarbituricacid method of Warren (Warren L., J. Biol. Chem. 234, 1971-1975, 1959)at 549 nm and 532 nm. In Steps 1, 2, and 7 explained below, thequantitation was performed by the same method after the reaction productwas passed through an AGX-2 ion exchange mini column. The amount of thesialic acid (nmol) released per hour was defined as 1 unit. When asynthetic substrate, 4-methylumbelliferyl N-acetylneuraminic acid(4MU-NeuAc) was used as the sialic acid-bound saccharide substrate,Triton X-100 was excluded from the reaction system, and released4-methylumbelliferone was quantitated by a fluorospectrophotometer.

In this example, the amount of enzyme protein was measured by theBradford method (a kit of Biorad Co. was used), or the BCA method (PieceChemical Co.). Further details of the measuring methods are found in theprevious report (Miyagi and Tsuiki, J. Biol. Chem. 260, 6710-6716,1985).

(2) Solubilization and Purification of Bovine Membrane-bound Sialidase

The whole procedure described below was carried out at 4° C. Bovinebrains obtained from a slaughterhouse were freezed at −80° C. until theywere used.

200 g of bovine brain was added 9 volumes of 0.32 M sucrose, 1 mM DTT(dithiothreitol), 1 mM EDTA, and 0.1 mM PMSF (phenylmethylsulfonylfluoride), homogenized by a glass Teflon homogenizer, and centrifuged at1000×g for 10 minute. The resulting supernatant was further centrifugedat 30,000×g for 1 hour. After the centrifucation, the resultingprecipitate fraction (Step 1) was suspended in Buffer A (20 mM potassiumphosphate, pH 6.8, 0.1% Triton X-100, 1 mM EDTA, 1 mM DTT) containing180 ml of 0.1 mM PMSF, then added 5% deoxycholic acid, sufficientlyhomogenized, and centrifuged at 100,000×g for one hour to obtain asupernatant as a soluble fraction (Step 2).

The soluble fraction was applied to a DEAE-cellulose column (4.5×20 cm)equilibrated with Buffer A, washed, and eluted with Buffer A containing0.2 M NaCl to collect 15 ml-fractions (Step 3). The active fraction wasdialyzed against Buffer A, then applied to an Octyl-Sepharose column(2.5×7 cm) equilibrated with the same buffer, and eluted with a lineargradient (400 ml) of 0.1-0.4% Triton X-100 to collect 10-ml fractions(Step 4).

Then, the active fraction was applied to a Heparin-Sepharose column(1.5×1 cm), washed with Buffer A containing 0.25 M NaCl, and eluted witha 0.2-1 M NaCl linear gradient (200 ml) in Buffer A, and the activefraction was concentrated by ultrafiltration using a YM-10 membrane(Step 5).

Concentrated enzyme solution obtained from 3 times of the elution fromthe Heparin-Sepharose column was loaded on a Sephacryl S-200 column(Pharmacia, 1.5×2.5 cm.), and eluted with Buffer B (20 mM potassiumphosphate, pH 6.8, 0.04% Triton X-100, 1 mM EDTA, 1 mM DTT, 0.02 mMNeuAc2en [2-deoxy-2,3-dehydro-N-acetylneuraminic acid]) at a flow rateof 10 ml/h to collect 2-ml fractions (Step 6).

The active fraction from Step 6, which was diluted so that it shouldhave a Triton X-100 concentration of 0.02%, was applied to an RCA-lectinagarose column (1.5×2.5 cm) equilibrated with Buffer B in which only theconcentration of Triton X-100 was changed to 0.02%, washed with the samebuffer, and eluted with Buffer B containing 0.2 M lactose (Step 7). Thisactive fraction was applied to a MonoQ (HR 5/5) column (Pharmacia),eluted with a 0-0.5 M NaCl linear gradient in Buffer B, and stored at−20° C. (Step 8).

The fraction obtained from 3 times of the elution in Step 8(corresponding to 1.8 kg of the starting material) was loaded on anactivated thiol-Sepharose column (Pharmacia, 1.5×2 cm), and eluted withBuffer B containing 0.15 M NaCl and 10% glycerol, and then with Buffer Bcontaining 0.5 M NaCl and 10% glycerol, and eluted with Buffer Bcontaining 0.5 M NaCl and DTT at a concentration raised to 50 mM. Theactive fraction was concentrated with a MonoQ column as in Step 8 (Step9).

Finally, affinity column chromatography utilizing a syntheticganglioside GM3 [GSC-211, NeuAc-Gal-Glc-O(CH₂)₈NH₂] (Hasegawa A. et al.,J. Carbohydr. Chem., 9, 201-214, 1990) as a ligand was performed. Anaffinity column (0.7×3 cm) was prepared by allowing the GSC-211 tocouple to ECH-Sepharose (Pharmacia) in the presence ofN-ethyl-N′-(3′-dimethyl-aminopropyl)carbodiimide hydrochloride accordingto the instruction of the manufacturer. The enzyme fraction obtainedfrom Step 8 was loaded on the column equilibrated with Buffer C (10 mMpotassium phosphate, pH 6.8, 0.04% Triton X-100, 1 mM EDTA, 1 mM DTT,20% glycerol), and eluted with a 0-0.5 M NaCl concentration gradient inBuffer C to collect 1.5 ml-fractions (Step 10).

The purification process using 3.5 kg of bovine brains as the startmaterial was summarized in Table 1. By the procedure explained above,the sialidase activity was purified by more than 100,000 times from thebovine brain particulate fraction. The final sample was subjected toSDS-polyacrylamide gel electrophoresis according to the method ofLaemmli (Laemmli, U.K. Nature, 227, 680-685, 1970) to determine itspurity. As a result, while a weak band at 50k was observed other thanthe main 52k protein band, the staining density of the 52k protein wasparallel with the activity elution pattern from the affinity column, andin addition, this band was concentrated from Step 9 to 10. Therefore, itwas considered to be a sialidase enzyme protein.

TABLE 1 Total Purifi- amount of Total Specific cation proteins activityactivity degree Yield Step (mg) (Unit) (Unit/mg) (−fold) (%) Particulate36622 1666327 45.5 1 100 fraction Solubilized 23057 1616337 70.1 1.5 97fraction DEAE- 13440 1051740 78.3 1.7 63 cellulose Octyl- 1581  486000307 6.7 29 cellulose Heparin- 112  245520 2188 48 15 Sepharose Sephacryl4.51  44670 9926 218 2.6 S-200 RCA-lectin 0.521  25771 49464 1087 1.5agarose Mono Q 0.220  19590 89045 1957 1.2 Activated- thiol 0.0103 18660 1811650 39816 1.1 Sepharose Ganglioside- 0.0012   5773 4851260106621 0.34 Sepharose

(3) Physicochemical Properties of Bovine Membrane-bound Sialidase

The physicochemical properties of the enzyme investigated by using theaforementioned purified enzyme are shown below.

(i) Substrate Specificity

The results of investigations about the activity of the enzyme of thepresent invention for various substrates are shown in Table 2. Thenumerical values represent relative activity when the activity for theGD3-ganglioside is defined to be 100.

TABLE 2 Substrate Hydrolysis activity (%) Ganglioside GD3-ganglioside100 GD1a-ganglioside 56 GM3-ganglioside 62 GM2-ganglioside 3GM1-ganglioside 1 Synthetic ganglioside GSC-17 (α2-3) 110 GSC-61 (α2-6)44 Orosomucoid 2 Fetuin 2 Glycophorin 3 Ovine submaxillary gland mucin 3Bovine submaxillary gland mucin 0 α2−3 syalyllactose 11 4-MUNeuAC 25

The hydrolysis of GSC-17 (α2-3) occurred at a rate 2.5 times higher thanthat of the hydrolysys of GSC-61 (α2-6), and hence the enzyme isconsidered to be more likely to act on α2-3 linkage compared with α2-6linkage. Further, since it did not act on α2-3 syalyllactosecorresponding to the saccharide segment of the GM3 ganglioside, theceramide segment is indispensable for a substrate.

(ii) Optimum pH

4.7 to 5.0

(iii) Molecular Weight

About 65,000 as determined by sucrose density gradient centrifugation,

About 52,000 as determined by SDS-polyacrylamide gel electrophoresisunder reducing condition.

(iv) Inhibition, Activation etc.

A detergent is required for the activity. For example, it is highlyactive in the presence of 0.1 to 0.2% of Triton X-100.

Residual activities in the presence of various inhibitors are shown inTable 3. The numerical values each represent 100 minus (residualactivity (%) in the presence of inhibitor).

TABLE 3 Inhibitor Inhibition (%) CuCl₂(1 mM) 95 4-Hydroxy mercurybenzoate (50 μM) 92 2-Deoxy-2,3-dehydro-N- 45 acetylneuraminic acid (0.2mM)

(3) Peptide Sequencing

Because the above-obtained final product was obtained at a low yield,the enzyme fraction of Step 9 was prepared in a similar manner by using6 kg of bovine brains as the starting material, and subjected to peptideanalysis. The enzyme fraction was desalted by dialysis in the presenceof 0.1% PVP-40 (Sigma), concentrated with Centricon (Millipore),subjected to SDS-polyacrylamide gel electrophoresis as described above,and transferred to a PVDF membrane (Problott, Applied Biosystems). Thelocation of the enzymatic protein was confirmed by Ponceau S staining,and the corresponding part of the membrane was excised, and digestedwith lysyl endopeptidase and then with endoproteinase Asp-N. The productwas separated by high performance liquid chromatography.

The fractionated peptide was subjected to amino acid sequencing using apeptide sequencer (Shimazu PSQ1). The above microsequencing wasperformed according to the method of Iwamatsu et al. (Iwamatsu A. andYoshida-Kubomura N., J. Biochem. 120, 29-34, 1996). The obtainedsequences are represented in FIG. 1 and SEQ ID NOS: 5-9. SEQ ID NOS: 5-7are amino acid sequences of the fragments obtained by the endoproteinasedigestion, and SEQ ID NOS: 8 and 9 are amino acid sequences of thefragments obtained by the lysyl endopeptidase digestion. In SEQ ID NO:5, the 2-5th amino acids are indefinite, and it is highly possible thatthe 2nd amino acid should be Ala or Arg, the 3rd amino acid be Glu orGly, the 4th amino acid be Ile or Tyr, and the 5th amino acid be Leu orSer.

<2> cDNA Cloning of Bovine Brain Sialidase

Based on the amino acid sequence of the peptide of the purified enzyme,which had been determined as described above, 10 sense or antisensedegenerate primers of SEQ ID NOS: 10 (DN1-1S), 11 (DN1-1A), 12 (DN1-2S),13 (DN1-2A), 14 (DN2S), 15 (DN2A), 16 (DN3A), 17 (AP1A), 18 (AP3S), and19 (AP3A) were prepared (see FIG. 1). DN1, DN2, DN-3, AP-1, and AP-3 aredesignations of the peptides shown in FIG. 1, S means “sense”, and Ameans “antisense”. DN-1 represents a nucleotide sequence determined byassuming that the undefinite amino acids of DN-1 (the 2-5th amino acids)should be Ala, Glu, Ile, and Leu, respectively, and DN-2 represents anucleotide sequence determined by assuming that the undefinite aminoacids of DN-1 should be Arg, Gly, Tyr, and Ser, respectively.

Bovine brain total RNA was prepared by the acidguanidium-phenol-chloroform method (Chomczynski P. and Sacchi N., Anal.Biochem. 162, 156-159, 1987), and poly(A) ⁺RNA was purified byoligo(dT)-cellulose column chromatography. cDNA was prepared inaccordance with the previous report (Miyagi T. et al., J. Biol. Chem.268, 26435-26440, 1993) using the poly(A) ⁺RNA (1 mg) and reversetranscriptase (derived from Molony murine leukemia virus, BRL), andamplification by PCR utilizing the cDNA as a template was attempted.

The PCR reaction mixture (50 ml) had a composition of 50 mM KCl, 10 mMTris-HCl (pH 8.3), 1.5 mM MgCl₂, 0.01% of gelatin, 0.2 mM dNTPs (2 mMeach of dATP, dGTP, dCTP and dTTP), 0.5 mg of cDNA, and 1.5 units of Taqpolymerase (Ex Taq, Takara). The DNA amplification was performed by 40cycles of reactions of at 94° C. (0.5 minutes), 50° C. (1 minute) and72° C. (2 minutes), followed by extension reaction at 72° C. for 10minutes. The obtained 12 DNA amplification fragments were each subclonedin the SmaI site of Bluescript vector (Stratagene), and subjected to DNAsequencing by the dideoxy method (Sanger F. et al., Proc. Natl. Acad.Sci. USA, 74, 5463-5467, 1977).

As a result of examination for the validity of the amino acid sequencesdeduced from the nucleotide sequences of the segments corresponding tothe primers within the amplified DNA fragments, presence or absence of astop codon and the like, it was found that only a PCR product of 0.5 kbobtained with primers AP3S and DN2A fulfilled those requirements. Inaddition, two Asp-boxes (-Ser-Xaa-Asp-Xaa-Gly-Xaa-Thr-Trp- (SEQ ID NO:20)), which is a consensus sequence of sialidase, were found in thisfragment (amino acid numbers 131-138 and 205-212 in SEQ ID NO: 2), andthe deduced amino acid sequence exhibited 38% homology with respect tothe amino acid sequence of the cytoplasmic sialidase, which we hadpreviously isolated. However, it did not exhibit significant homologywith any other proteins.

Then, a bovine brain λgt10 library (Clontech) was screened by using theabove 0.5 kb cDNA as a probe. The cDNA was isotope-labeled with[α-³²P]dCTP using Random Primer DNA Labeling Kit (Takara), and phages(2×10⁶) were screened by plaque hybridization. The hybridization wascarried out by using a nylon membrane (Hybond N⁺, Amersham) according tothe instruction of the manufacturer. Among 15 positive clones, two ofpBB121 (1.45 kb) and pBB321 (2.8 kb) were estimated to contain the fullcoding region.

The nucleotide sequence of the insert of pBB321 (2.8 kb) and the aminoacid sequence deduced from it are shown as SEQ ID NOS: 1 and 2. It wasfound that four types of amino acid sequences obtained from the peptideof the purified product were contained in it, and as for DN-1, the 2ndamino acid of DN 1-1 was A R. Moreover, because it was found that thesame sequence was contained in bovine keratin based on protein databasesearch, although it was not found for the AP-1 sequence, it is highlypossible that it originated from contamination of keratin in the enzymefraction used for the peptide sequencing.

In addition to the above-mentioned two Asp-boxes, another Asp-box wasfurther found on their 3′ side (the amino acid numbers 256-263 in SEQ IDNO: 2). A hydrophobic sequence considered to be a transmembrane domainwas found between the two Asp-boxes (the amino acid numbers 174-194 inSEQ ID NO: 2), and a glycosylation site was found on its 3′ side (theamino acid number 349 in SEQ ID NO: 2). Because the enzyme has thecharacteristic that it is bound to RCA lectin as used in thepurification procedure, it is considered that a saccharide chainactually attached to this site. The molecular weight of the proteincalculated from the 428 amino acids is 48,000, and if one saccharidechain is attached, the actual value will become around 50,000, and it isnot contradictory to the value determined for the above purified productby SDS-polyacrylamide gel electrophoresis.

<3> Transient Expression of Sialidase cDNA in COS Cell

The coding region of pBB121 (1.45 kb) was amplified by PCR using 5′sense primer (SEQ ID NO: 21) which was added an EcoRI site and 3′antisense primer (SEQ ID NO: 22), and the obtained DNA fragment waspurified by agarose electrophoresis. This product was ligated to theEcoRI site of SRα promoter high expression vector pME18S (provided byDr. Kazuo Maruyama, Medical Department, Tokyo Medical and DentalUniversity) having SV40 replication origin (pME18S-mSD), and introducedinto COS-7 cells by electroporation to attempt its transient expression.Forty μg of pME18S or pME18S-mSD was added to COS-7 cells (10⁶) culturedin DMEM containing 10% FBS (fetal bovine serum) in the logarithmicgrowth phase, left at room temperature for 10 minutes, applied withelectric pulses at 250 V and 950 μFD, left at room temperature again for10 minutes, and then returned to the culture.

The cells after the culture of 48 hours were collected. After the bloodserum components were removed with PBS, the cells were added 9 volumesof PBS, and disrupted by ultrasonication for 10 seconds. The disruptedcell suspension was centrifuged under cooling at 1,000×g for 10 minutes,and the resulting supernatant was used as a homogenate. Sialidaseactivity in the homogenate of the transfectants was measured by usinggangliosides as the substrate in the presence of Triton X-100 (0.1%).

The specific activity of control cells having only the vector and thecells that were introduced with pME18S-mSD was 23.4 units/mg protein and844.5 units/mg protein, respectively. Thus, the cells introduced withpME18S-mSD exhibited the activity 36 times higher than that of thecontrol cells. However, increase of the activity for hydrolyzing4MU-sialic acid was not observed at all. This result confirmed theresults of the previous characterization of the purified product of thebovine brain enzyme, i.e., the expressed sialidase substantiallyspecifically acted on gangliosides, and hardly acted on syntheticsubstrates such as 4MU-sialic acid.

Further, it was investigated whether the expressed sialidase localizedin plasma membrane or not by using Percoll (Pharmacia) concentrationgradient centrifugation. In accordance with a previous report (Sagawa J.et al., J. Biochem. 107, 452-456, 1990), the homogenate was overlaid on40% Percoll containing 0.25 M sucrose, centrifuged at 48,000×g for 40minutes, and fractionated, and the sialidase activity was measured.Ganglioside sialidase activity was detected at the same location as theactivity distribution of 5′-nucleotidase or alkali phosphatase, whichare marker enzymes of plasma membrane, and thus the localization of theexpressed sialidase in plasma membrane was confirmed.

<4> cDNA Cloning of Human-derived Ganglioside Sialidase

When the primary structure of the bovine brain sialidase was comparedwith the previously isolated cytoplasmic sialidase (Miyagi T. et al., J.Biol. Chem., 268, 26435-26440, 1993), it was found that they contained asequence well conserved in them (FIG. 2). Therefore, one set of primerswas prepared based on this amino acid sequence (SEQ ID NOS: 23 and 24).In the amino acid sequences shown in FIG. 2, the partial sequence ofcDNA for bovine brain sialidase (BBmSD) corresponds to the amino acidnumbers 49-209 of SEQ ID NO: 2. The rat skeletal muscle cytoplasmicsialidase (RMcSD) corresponds to the amino acid numbers 1-240 in theamino acid sequence of the sialidase.

Human brain cDNA and human kidney cDNA were prepared under the samecondition as the case of the bovine enzyme, and PCR was performed byusing them as the template. The amplified DNA fragment of 0.25 kb wassubcloned, and the DNA sequence was determined. One Asp-box was found inthis cDNA. A human brain λgt10 cDNA library and human kidney λgt10 cDNAlibrary (Clontech) were screened by using the above DNA as a probe. Byscreening 8×10⁵ plaques for human brain and 1×10⁶ plaques for humankidney, three positive clones (pHB82, pHB85, and pHB95) and one positiveclone (pHK65) were obtained, respectively.

When these DNA sequences were investigated after subcloning, anoverlapped portion of 1 kb was found in all of them. Nucleotidesequences obtained from pHB95 containing the substantially whole codingregion and pHK65 containing 3′ end non-coding region of 1 kb, anddeduced amino acid sequences therefor are shown as SEQ ID NOS: 3 and 4.They exhibited high homology with the sequences for bovine brain enzyme,i.e., 81% (87% for only the coding region) on the nucleotide level, and82% on the amino acid level.

In SEQ ID NO: 4, the transmembrane domain corresponds to the amino acidnumbers 174-194, the glycosylation site to the amino acid number 348,and the Asp-boxes to the amino acid numbers 131-138, 205-212, and256-263.

When the expression status was investigated in various human tissues byNorthern blotting using the 1.5 kb insert of pHB95 as the probe, highexpression of mRNA of about 4 kb was observed in skeletal muscles, andmRNA of the same size was also detected in brain, liver and the like.

Industrial Availability

The present invention provides sialidase localized in plasma membraneand DNA coding for it. The sialidase of the present invention differsfrom sialidases known so far in that it mainly localizes in plasmamembrane, and specifically hydrolyze gangliosides.

24 1 3003 DNA Bos primigenius taurus CDS (259)..(1542) 1 ggagcttcctggacttcctt tcctaacggc tgttttcggc ttccccaatc tgtcagcccc 60 gccgccagcctctcgatgtc tctgtcgccg tgtttcttca cttttcgtgg tttgtgtccg 120 cgtccgcagtttctctcctg ccctcgtctc cagggcttga tcattctcca gggcttcagt 180 gtcggagacgtgagtgcttg acccagcgcc cagatcagcc cgagagagat ggaggagccg 240 gggttccctgcagaggtc atg gaa gaa gtg aca tca tgc tcc ttc agc agc 291 Met Glu Glu ValThr Ser Cys Ser Phe Ser Ser 1 5 10 cct ctg ttc cag cag gag gac aag agaggg gtc acc tac cgg atc cca 339 Pro Leu Phe Gln Gln Glu Asp Lys Arg GlyVal Thr Tyr Arg Ile Pro 15 20 25 gcc ctg atc tac gtg ccc cct gcc cac accttc ctg gcc ttt gca gag 387 Ala Leu Ile Tyr Val Pro Pro Ala His Thr PheLeu Ala Phe Ala Glu 30 35 40 aag cgc tcc tcg agc aag gat gag gat gct ctccac ctg gtg ctg agg 435 Lys Arg Ser Ser Ser Lys Asp Glu Asp Ala Leu HisLeu Val Leu Arg 45 50 55 cga gga tta agg act ggg caa tca gta cag tgg gaaccc ctg aag tcc 483 Arg Gly Leu Arg Thr Gly Gln Ser Val Gln Trp Glu ProLeu Lys Ser 60 65 70 75 ctg atg aaa gcc acg tta cct gga cac cgg acc atgaac ccc tgt cct 531 Leu Met Lys Ala Thr Leu Pro Gly His Arg Thr Met AsnPro Cys Pro 80 85 90 gtg tgg gag cgg aag agt ggc tac gtg tac ctg ttc ttcatc tgt gtg 579 Val Trp Glu Arg Lys Ser Gly Tyr Val Tyr Leu Phe Phe IleCys Val 95 100 105 caa ggc cat gtc acc gag cgt caa cag att atg tca ggcagg aac cct 627 Gln Gly His Val Thr Glu Arg Gln Gln Ile Met Ser Gly ArgAsn Pro 110 115 120 gca cgc ctc tgc ttc ata tgc agc cag gat gct ggc tattca tgg agt 675 Ala Arg Leu Cys Phe Ile Cys Ser Gln Asp Ala Gly Tyr SerTrp Ser 125 130 135 gat gtg agg gac ctg act gag gag gtc att ggc cca gaggtg aca cac 723 Asp Val Arg Asp Leu Thr Glu Glu Val Ile Gly Pro Glu ValThr His 140 145 150 155 tgg gcc act ttt gct gtg ggg cca ggt cat ggc atccag ctg cag tcg 771 Trp Ala Thr Phe Ala Val Gly Pro Gly His Gly Ile GlnLeu Gln Ser 160 165 170 ggg agg ctc atc atc cct gca tat gcc tac tac atcccg ttc tgg ttc 819 Gly Arg Leu Ile Ile Pro Ala Tyr Ala Tyr Tyr Ile ProPhe Trp Phe 175 180 185 ttt tgc ttt cgg ctg cca tat aga gct agg cct cattcc ctg atg atc 867 Phe Cys Phe Arg Leu Pro Tyr Arg Ala Arg Pro His SerLeu Met Ile 190 195 200 tat agc gat gac cta gga gcc aca tgg cac cat ggcagg ctt atc aag 915 Tyr Ser Asp Asp Leu Gly Ala Thr Trp His His Gly ArgLeu Ile Lys 205 210 215 ccc atg gtg aca gtg gaa tgt gaa gtg gca gag gtgatc ggg aag gcc 963 Pro Met Val Thr Val Glu Cys Glu Val Ala Glu Val IleGly Lys Ala 220 225 230 235 ggc cac cct gtg ctg tat tgc agt gcc cgg acacca aac agg cac cgg 1011 Gly His Pro Val Leu Tyr Cys Ser Ala Arg Thr ProAsn Arg His Arg 240 245 250 gca gag gcc ctc agc att gac cat ggt gaa tgcttt cag aaa cca gtc 1059 Ala Glu Ala Leu Ser Ile Asp His Gly Glu Cys PheGln Lys Pro Val 255 260 265 ctg agc cat cag ctc tgt gag ccc cct cat ggctgt caa ggc agt gtg 1107 Leu Ser His Gln Leu Cys Glu Pro Pro His Gly CysGln Gly Ser Val 270 275 280 gtg agt ttc tgt ccc ctg gag atc cca ggt ggatgc cag gat ctt gct 1155 Val Ser Phe Cys Pro Leu Glu Ile Pro Gly Gly CysGln Asp Leu Ala 285 290 295 ggc gaa gat gca cct gcc att cag cag agt cctctg ctg tgc agc tca 1203 Gly Glu Asp Ala Pro Ala Ile Gln Gln Ser Pro LeuLeu Cys Ser Ser 300 305 310 315 gtg aga cca gag ccg gaa gct gga acc ctgtca gaa tca tgg ctc ttg 1251 Val Arg Pro Glu Pro Glu Ala Gly Thr Leu SerGlu Ser Trp Leu Leu 320 325 330 tac tca cac cca acc aat aag aaa cgg agggtc gat cta ggc atc tac 1299 Tyr Ser His Pro Thr Asn Lys Lys Arg Arg ValAsp Leu Gly Ile Tyr 335 340 345 ctc aac cag agc ccc ttg gag gct gcc tgctgg tcc cgc ccc tgg atc 1347 Leu Asn Gln Ser Pro Leu Glu Ala Ala Cys TrpSer Arg Pro Trp Ile 350 355 360 ttg cac tgc ggg ccc tgt ggg tac tct gatttg gct gct ctg gag aat 1395 Leu His Cys Gly Pro Cys Gly Tyr Ser Asp LeuAla Ala Leu Glu Asn 365 370 375 gag ggc ttg ttt ggg tgt ttg ttt gaa tgtggg acc aag cag gag tgt 1443 Glu Gly Leu Phe Gly Cys Leu Phe Glu Cys GlyThr Lys Gln Glu Cys 380 385 390 395 gag cag att gcc ttc cgc ctg ttt acagac cga gag atc ctg agc cac 1491 Glu Gln Ile Ala Phe Arg Leu Phe Thr AspArg Glu Ile Leu Ser His 400 405 410 gtg caa ggg gac tgc tcc acc cct ggtatg aac tct gag cca agt aaa 1539 Val Gln Gly Asp Cys Ser Thr Pro Gly MetAsn Ser Glu Pro Ser Lys 415 420 425 aag taattcgctt aggacccaac tttgcatagaaggctaccgt agaaggcagt 1592 Lys cacagccagg acagtggagg ccaggataacagaggttact gaagtctgca gagaaacaaa 1652 acacctaata ttctgctccc tacctgttttcacttctcat tctccagaga acaaaatgaa 1712 catcttgcca tagctactgc attcaaaagagcactgaacg gtgagctgag agactatgat 1772 gtcatcttgg ctcttccact ggcttgctttgggaccttgg acatgtcacc tgtactctct 1832 gggcctcagg tctccatctg taaaaggagagggtcggatc tctgatttct cttcttccca 1892 tccctaggaa aggcagtgtg cctgcatgccccctgatcag caagtcctgg ctgtatgtag 1952 gactcttatc tcaaaggcag gctccgcttttcaaatgact tgccactcat ccaagtataa 2012 ggttacaagc aggtgtcata gcacaaaggaagatgtaggt ggcctgtttt gtttttaata 2072 acaaaagcac ttacatcctt ctgattatgcacgaagctct acagactcac tgttctagag 2132 gaatcgggcc aagcagcaga attataggtcacttaccttc tccagcttta cagctctgct 2192 ccacctttcc ttccttgtcc agaaagcattacctctgaag gagaaaatga gatgctcaat 2252 gtcagtgatc ttcaataatg gtacttaatgtttctgctgg catgactcct atgagagatg 2312 aacttgaagt tcatttatta ggatagttattgatgagaaa tgaacatggg ttaggacttc 2372 aaagcatcgg acaaaacttt ctgctattgctgctctcaag gagttcacag tttagggggc 2432 tagaagaggg ataaaattga agaaaataaatgtagctggg gggatagttt atagatattg 2492 ggctctaagt gggagtgata gtagctgctgatggtattat tttaattgta tcttaattgt 2552 gcctggagtc atctgcccca gaacttgtccaagctgctgt ttgtttttct cagaatgttg 2612 ttttcactca gccttcttta atggagacagtcgtcaccat tcagaaggtc tctggactca 2672 aaaacctctg aatcaagcat atttgttcagacctactgaa atttggacca tctctactat 2732 tagtgaagtg tagagatgct tctttatctaatagatttgg gataaacttt gacattgctg 2792 gttctcagat gatagcagat ggttgctcttattttagatc atttcctcca taagcctttt 2852 actgtgacag atactcttat tgtgagagctaccttttttg tccctatttt tggaggataa 2912 tgccttaaac aggcagcagg taaatatatttggtgctgag taatgaccct ggagagtaag 2972 tcgttgtcgt ggaacacagc ctagaaagtg g3003 2 428 PRT Bos primigenius 2 Met Glu Glu Val Thr Ser Cys Ser Phe SerSer Pro Leu Phe Gln Gln 1 5 10 15 Glu Asp Lys Arg Gly Val Thr Tyr ArgIle Pro Ala Leu Ile Tyr Val 20 25 30 Pro Pro Ala His Thr Phe Leu Ala PheAla Glu Lys Arg Ser Ser Ser 35 40 45 Lys Asp Glu Asp Ala Leu His Leu ValLeu Arg Arg Gly Leu Arg Thr 50 55 60 Gly Gln Ser Val Gln Trp Glu Pro LeuLys Ser Leu Met Lys Ala Thr 65 70 75 80 Leu Pro Gly His Arg Thr Met AsnPro Cys Pro Val Trp Glu Arg Lys 85 90 95 Ser Gly Tyr Val Tyr Leu Phe PheIle Cys Val Gln Gly His Val Thr 100 105 110 Glu Arg Gln Gln Ile Met SerGly Arg Asn Pro Ala Arg Leu Cys Phe 115 120 125 Ile Cys Ser Gln Asp AlaGly Tyr Ser Trp Ser Asp Val Arg Asp Leu 130 135 140 Thr Glu Glu Val IleGly Pro Glu Val Thr His Trp Ala Thr Phe Ala 145 150 155 160 Val Gly ProGly His Gly Ile Gln Leu Gln Ser Gly Arg Leu Ile Ile 165 170 175 Pro AlaTyr Ala Tyr Tyr Ile Pro Phe Trp Phe Phe Cys Phe Arg Leu 180 185 190 ProTyr Arg Ala Arg Pro His Ser Leu Met Ile Tyr Ser Asp Asp Leu 195 200 205Gly Ala Thr Trp His His Gly Arg Leu Ile Lys Pro Met Val Thr Val 210 215220 Glu Cys Glu Val Ala Glu Val Ile Gly Lys Ala Gly His Pro Val Leu 225230 235 240 Tyr Cys Ser Ala Arg Thr Pro Asn Arg His Arg Ala Glu Ala LeuSer 245 250 255 Ile Asp His Gly Glu Cys Phe Gln Lys Pro Val Leu Ser HisGln Leu 260 265 270 Cys Glu Pro Pro His Gly Cys Gln Gly Ser Val Val SerPhe Cys Pro 275 280 285 Leu Glu Ile Pro Gly Gly Cys Gln Asp Leu Ala GlyGlu Asp Ala Pro 290 295 300 Ala Ile Gln Gln Ser Pro Leu Leu Cys Ser SerVal Arg Pro Glu Pro 305 310 315 320 Glu Ala Gly Thr Leu Ser Glu Ser TrpLeu Leu Tyr Ser His Pro Thr 325 330 335 Asn Lys Lys Arg Arg Val Asp LeuGly Ile Tyr Leu Asn Gln Ser Pro 340 345 350 Leu Glu Ala Ala Cys Trp SerArg Pro Trp Ile Leu His Cys Gly Pro 355 360 365 Cys Gly Tyr Ser Asp LeuAla Ala Leu Glu Asn Glu Gly Leu Phe Gly 370 375 380 Cys Leu Phe Glu CysGly Thr Lys Gln Glu Cys Glu Gln Ile Ala Phe 385 390 395 400 Arg Leu PheThr Asp Arg Glu Ile Leu Ser His Val Gln Gly Asp Cys 405 410 415 Ser ThrPro Gly Met Asn Ser Glu Pro Ser Lys Lys 420 425 3 1892 DNA Homo sapiensCDS (11)..(1294) 3 tgcagaggtc atg gaa gaa gtg aca aca tgc tcc ttc aacagc cct ctg 49 Met Glu Glu Val Thr Thr Cys Ser Phe Asn Ser Pro Leu 1 510 ttc cgg cag gaa gat gac aga ggg att acc tac cgg atc cca gcc ctg 97Phe Arg Gln Glu Asp Asp Arg Gly Ile Thr Tyr Arg Ile Pro Ala Leu 15 20 25ctc tac ata ccc ccc acc cac acc ttc ctg gcc ttt gca gag aag cgt 145 LeuTyr Ile Pro Pro Thr His Thr Phe Leu Ala Phe Ala Glu Lys Arg 30 35 40 45tcc acg agg aga gat gag gat gct ctc cac ctg gtg ctg agg cga ggg 193 SerThr Arg Arg Asp Glu Asp Ala Leu His Leu Val Leu Arg Arg Gly 50 55 60 ttgagg att ggg cag ttg gta cag tgg ggg ccc ctg aag cca ctg atg 241 Leu ArgIle Gly Gln Leu Val Gln Trp Gly Pro Leu Lys Pro Leu Met 65 70 75 gaa gccaca cta ccg ggg cat cgg acc atg aac ccc tgt cct gta tgg 289 Glu Ala ThrLeu Pro Gly His Arg Thr Met Asn Pro Cys Pro Val Trp 80 85 90 gag cag aagagt ggt tgt gtg ttc ctg ttc ttc atc tgt gtg cgg ggc 337 Glu Gln Lys SerGly Cys Val Phe Leu Phe Phe Ile Cys Val Arg Gly 95 100 105 cat gtc acagag cgt caa cag att gtg tca ggc agg aat gct gcc cgc 385 His Val Thr GluArg Gln Gln Ile Val Ser Gly Arg Asn Ala Ala Arg 110 115 120 125 ctt tgcttc atc tac agt cag gat gct gga tgt tca tgg agt gag gtg 433 Leu Cys PheIle Tyr Ser Gln Asp Ala Gly Cys Ser Trp Ser Glu Val 130 135 140 agg gacttg act gag gag gtc att ggc tca gag ctg aag cac tgg gcc 481 Arg Asp LeuThr Glu Glu Val Ile Gly Ser Glu Leu Lys His Trp Ala 145 150 155 aca tttgct gtg ggc cca ggt cat ggc atc cag ctg cag tca ggg aga 529 Thr Phe AlaVal Gly Pro Gly His Gly Ile Gln Leu Gln Ser Gly Arg 160 165 170 ctg gtcatc cct gcg tat acc tac tac atc cct tcc tgg ttc ttt tgc 577 Leu Val IlePro Ala Tyr Thr Tyr Tyr Ile Pro Ser Trp Phe Phe Cys 175 180 185 ttc cagcta cca tgt aaa acc agg cct cat tct ctg atg atc tac agt 625 Phe Gln LeuPro Cys Lys Thr Arg Pro His Ser Leu Met Ile Tyr Ser 190 195 200 205 gatgac cta ggg gtc aca tgg cac cat ggt aga ctc att agg ccc atg 673 Asp AspLeu Gly Val Thr Trp His His Gly Arg Leu Ile Arg Pro Met 210 215 220 gttaca gta gaa tgt gaa gtg gca gag gtg act ggg agg gct ggc cac 721 Val ThrVal Glu Cys Glu Val Ala Glu Val Thr Gly Arg Ala Gly His 225 230 235 cctgtg cta tat tgc agt gcc cgg aca cca aac agg tgc cgg gca gag 769 Pro ValLeu Tyr Cys Ser Ala Arg Thr Pro Asn Arg Cys Arg Ala Glu 240 245 250 gcgctc agc act gac cat ggt gaa ggc ttt cag aga ctg gcc ctg agt 817 Ala LeuSer Thr Asp His Gly Glu Gly Phe Gln Arg Leu Ala Leu Ser 255 260 265 cgacag ctc tgt gag ccc cca cat ggt tgc caa ggg agt gtg gta agt 865 Arg GlnLeu Cys Glu Pro Pro His Gly Cys Gln Gly Ser Val Val Ser 270 275 280 285ttc cgg ccc ctg gag atc cca cat agg tgc cag gac tct agc agc aaa 913 PheArg Pro Leu Glu Ile Pro His Arg Cys Gln Asp Ser Ser Ser Lys 290 295 300gat gca ccc acc att cag cag agc tct cca ggc agt tca ctg agg ctg 961 AspAla Pro Thr Ile Gln Gln Ser Ser Pro Gly Ser Ser Leu Arg Leu 305 310 315gag gag gaa gct gga aca ccg tca gaa tca tgg ctc ttg tac tca cac 1009 GluGlu Glu Ala Gly Thr Pro Ser Glu Ser Trp Leu Leu Tyr Ser His 320 325 330cca acc agt agg aaa cag agg gtt gac cta ggt atc tat ctc aac cag 1057 ProThr Ser Arg Lys Gln Arg Val Asp Leu Gly Ile Tyr Leu Asn Gln 335 340 345acc ccc ttg gag gct gcc tgc tgg tcc cgc ccc tgg atc ttg cac tgt 1105 ThrPro Leu Glu Ala Ala Cys Trp Ser Arg Pro Trp Ile Leu His Cys 350 355 360365 ggg ccc tgt ggc tac tct gat ctg gct gct ctg gag gag gag ggc ttg 1153Gly Pro Cys Gly Tyr Ser Asp Leu Ala Ala Leu Glu Glu Glu Gly Leu 370 375380 ttt ggg tgt ttg ttt gaa tgt ggg acc aag caa gag tgt gag cag att 1201Phe Gly Cys Leu Phe Glu Cys Gly Thr Lys Gln Glu Cys Glu Gln Ile 385 390395 gcc ttc cgc ctg ttt aca cac cgg gag atc ctg agt cac ctg cag ggg 1249Ala Phe Arg Leu Phe Thr His Arg Glu Ile Leu Ser His Leu Gln Gly 400 405410 gac tgc acc agc cct ggt agg aac cca agc caa ttc aaa agc aat 1294 AspCys Thr Ser Pro Gly Arg Asn Pro Ser Gln Phe Lys Ser Asn 415 420 425taattggctt aggacccaat ttccatagat gcaaatggca gttacagaca ggttaacaga 1354agctactgaa gtctacagat aatcaaaaaa cttaatattc tgttccctac cttttttcac 1414ttttcctcct ccaaagagca aaatgaaaat tttgccttag ctactgcagt ggaaagagca 1474ctgaactagg agttggaaga caaggatgtg gtcctggctc tgcactggct tgcttttgga 1534ccttggatgt gtcacctgaa ctctctggac ctcaggtttc catctgtaaa atgagagtat 1594tggttctaag atttctcatc ttctcatccc taggacaagc atagtgcctg catgcttcat 1654gatcagtaag tcctggctgc ataaaggact ctgatgtcaa aatggaaacc aggggactta 1714ccttttcaca tgacttaccc ctcatccgag tgtgaggtta caagcaggtg tcatggcagg 1774aaggaagacc agatctgtat gatttgttcc atttttaata acaaaaatat ccacaccctt 1834ttaataatgc tcagagttct gtaggctctc tatcctagag gaattgagca aaacagcc 1892 4428 PRT Homo sapiens 4 Met Glu Glu Val Thr Thr Cys Ser Phe Asn Ser ProLeu Phe Arg Gln 1 5 10 15 Glu Asp Asp Arg Gly Ile Thr Tyr Arg Ile ProAla Leu Leu Tyr Ile 20 25 30 Pro Pro Thr His Thr Phe Leu Ala Phe Ala GluLys Arg Ser Thr Arg 35 40 45 Arg Asp Glu Asp Ala Leu His Leu Val Leu ArgArg Gly Leu Arg Ile 50 55 60 Gly Gln Leu Val Gln Trp Gly Pro Leu Lys ProLeu Met Glu Ala Thr 65 70 75 80 Leu Pro Gly His Arg Thr Met Asn Pro CysPro Val Trp Glu Gln Lys 85 90 95 Ser Gly Cys Val Phe Leu Phe Phe Ile CysVal Arg Gly His Val Thr 100 105 110 Glu Arg Gln Gln Ile Val Ser Gly ArgAsn Ala Ala Arg Leu Cys Phe 115 120 125 Ile Tyr Ser Gln Asp Ala Gly CysSer Trp Ser Glu Val Arg Asp Leu 130 135 140 Thr Glu Glu Val Ile Gly SerGlu Leu Lys His Trp Ala Thr Phe Ala 145 150 155 160 Val Gly Pro Gly HisGly Ile Gln Leu Gln Ser Gly Arg Leu Val Ile 165 170 175 Pro Ala Tyr ThrTyr Tyr Ile Pro Ser Trp Phe Phe Cys Phe Gln Leu 180 185 190 Pro Cys LysThr Arg Pro His Ser Leu Met Ile Tyr Ser Asp Asp Leu 195 200 205 Gly ValThr Trp His His Gly Arg Leu Ile Arg Pro Met Val Thr Val 210 215 220 GluCys Glu Val Ala Glu Val Thr Gly Arg Ala Gly His Pro Val Leu 225 230 235240 Tyr Cys Ser Ala Arg Thr Pro Asn Arg Cys Arg Ala Glu Ala Leu Ser 245250 255 Thr Asp His Gly Glu Gly Phe Gln Arg Leu Ala Leu Ser Arg Gln Leu260 265 270 Cys Glu Pro Pro His Gly Cys Gln Gly Ser Val Val Ser Phe ArgPro 275 280 285 Leu Glu Ile Pro His Arg Cys Gln Asp Ser Ser Ser Lys AspAla Pro 290 295 300 Thr Ile Gln Gln Ser Ser Pro Gly Ser Ser Leu Arg LeuGlu Glu Glu 305 310 315 320 Ala Gly Thr Pro Ser Glu Ser Trp Leu Leu TyrSer His Pro Thr Ser 325 330 335 Arg Lys Gln Arg Val Asp Leu Gly Ile TyrLeu Asn Gln Thr Pro Leu 340 345 350 Glu Ala Ala Cys Trp Ser Arg Pro TrpIle Leu His Cys Gly Pro Cys 355 360 365 Gly Tyr Ser Asp Leu Ala Ala LeuGlu Glu Glu Gly Leu Phe Gly Cys 370 375 380 Leu Phe Glu Cys Gly Thr LysGln Glu Cys Glu Gln Ile Ala Phe Arg 385 390 395 400 Leu Phe Thr His ArgGlu Ile Leu Ser His Leu Gln Gly Asp Cys Thr 405 410 415 Ser Pro Gly ArgAsn Pro Ser Gln Phe Lys Ser Asn 420 425 5 10 PRT Bos primigenius taurusUNSURE (2) Xaa=ala or arg 5 Asp Xaa Xaa Xaa Xaa Ser His Val Gln Gly 1 510 6 5 PRT Bos primigenius taurus 6 Asp Asp Leu Gly Ala 1 5 7 5 PRT Bosprimigenius taurus 7 Glu Glu Val Thr Ser 1 5 8 5 PRT Bos primigeniustaurus 8 Lys Tyr Glu Glu Leu 1 5 9 9 PRT Bos primigenius taurus 9 LysAsp Glu Asp Ala Leu His Leu Val 1 5 10 26 DNA Artificial SequenceDescription of Artificial Sequencesynthetic oligonucleotide 10gaygcngara tyctnwnnca ygtnca 26 11 29 DNA Artificial SequenceDescription of Artificial Sequencesynthetic oligonucleotide 11ccctgnacrt gnnwnagrat tycngcrtc 29 12 29 DNA Artificial SequenceDescription of Artificial Sequencesynthetic oligonucleotide 12gayngnggnt aynsnwnnca ygtncaggg 29 13 29 DNA Artificial SequenceDescription of Artificial Sequencesynthetic oligonucleotide 13ccctgnacrt gnnwnsnrta nccncnrtc 29 14 14 DNA Artificial SequenceDescription of Artificial Sequencesynthetic oligonucleotide 14gaygayctng gngc 14 15 14 DNA Artificial Sequence Description ofArtificial Sequencesynthetic oligonucleotide 15 gcnccnagrt crtc 14 16 15DNA Artificial Sequence Description of Artificial Sequencesyntheticoligonucleotide 16 nsnngtnacy tcytc 15 17 15 DNA Artificial SequenceDescription of Artificial Sequencesynthetic oligonucleotide 17nanytcytcr taytt 15 18 26 DNA Artificial Sequence Description ofArtificial Sequencesynthetic oligonucleotide 18 aargaygarg aygcnctncayctngt 26 19 23 DNA Artificial Sequence Description of ArtificialSequencesynthetic oligonucleotide 19 acnagrtgna gngcrtcytc rtc 23 20 8PRT Unknown Description of Unknown Organismconsensus sequence 20 Ser XaaAsp Xaa Gly Xaa Thr Trp 1 5 21 30 DNA Artificial Sequence Description ofArtificial Sequencesynthetic oligonucleotide 21 cccgaattcg tcatggaagaagtgacatca 30 22 30 DNA Artificial Sequence Description of ArtificialSequencesynthetic oligonucleotide 22 cccgaattct tactttttac ttggctcaga 3023 27 DNA Artificial Sequence Description of ArtificialSequencesynthetic oligonucleotide 23 ggacaccgga ccatgaaccc ctgtcct 27 2426 DNA Artificial Sequence Description of Artificial Sequencesyntheticoligonucleotide 24 cctggcccca cagcaaaagt ggccca 26

What is claimed is:
 1. An isolated DNA encoding a protein comprising anamino acid sequence of SEQ ID NO:
 2. 2. The DNA according to claim 1,wherein the protein includes substitution, deletion, or insertion of oneor several amino acid residues, and exhibits activity to remove a sialicacid residue from a non-reducing terminal of ganglioside.
 3. A vectorcomprising the DNA of claim
 1. 4. An isolated DNA encoding for a proteincomprising an amino acid sequence of SEQ ID NO:
 4. 5. The DNA accordingto claim 4, wherein the protein includes substitution, deletion, orinsertion of one or several amino acid residues, and exhibits activityto remove a sialic acid residue from a non-reducing terminal ofganglioside.
 6. A vector comprising the DNA of claim
 4. 7. The DNAaccording to claim 1 which has a nucleotide sequence of SEQ ID NO:
 1. 8.The DNA according to claim 4 which has a nucleotide sequence of SEQ IDNO:
 3. 9. A vector comprising the DNA of claim
 7. 10. A vectorcomprising the DNA of claim 8.